Barley yellow dwarf virus (BYDV) is a plus-stranded RNA virus that belongs to the genus Luteovirus of the Luteoviridae family. It is a very widespread pathogen of wheat, barley and oats. Plant viruses, whose genomes are usually smaller than animal viruses, are easier to culture and use in a variety of in vitro and in vivo studies, giving plant viruses the advantage over animal viruses for studying replication, transcription, and translation control events. BYDV utilizes an assortment of strategies to express its genes and serves as a unique model to study novel transcriptional and translational control events. Like many (+) sense RNA viruses, BYDV deploys subgenomic (sg) RNAs to express its 3'-proximal genes. This strategy allows downstream ORFs to be positioned at the 5' end of the subgenomic mRNA, making them available to ribosomes for protein synthesis. Since transcription, replication and translation are all connected during the actual infection of the host, the long term goal is to develop a model that will explain the role of sgRNA synthesis in translation control. Understanding the mechanisms of how subgenomic RNAs are synthesized is relevant to many human pathogens such as alphaviruses and coronaviruses. The proposed research project centers on determining the molecular mechanisms involved in the synthesis of subgenomic RNAs in BYDV, particularly the synthesis of subgenomic RNAs 2 and 3. The specific aims of this proposal are to investigate the regulation and mechanism(s) of subgenomic RNA synthesis by (1) determining the essential primary and secondary structures required for synthesis of subgenomic RNAs 2 and 3 (2) investigating possible trans-replication of sgRNAs 2 and 3 (3) determining the roles of sgRNA2 and sgRNA3, including mutants, in virus infection of whole plants.